Gastric bypass surgery in lean adolescent mice prevents diet-induced obesity later in life.

Gastric bypass surgery is the most effective treatment and is often the only option for subjects with severe obesity. However, investigation of critical molecular mechanisms involved has been hindered by confounding of specific effects of surgery and side effects associated with acute surgical trauma. Here, we dissociate the two components by carrying out surgery in the lean state and testing its effectiveness to prevent diet-induced obesity later in life. Body weight and composition of female mice with RYGB performed at 6 weeks of age were not significantly different from sham-operated and age-matched non-surgical mice at the time of high-fat diet exposure 12 weeks after surgery. These female mice were completely protected from high-fat diet-induced obesity and accompanying metabolic impairments for up to 50 weeks. Similar effects were seen in male mice subjected to RYGB at 5-6 weeks, although growth was slightly inhibited and protection from diet-induced obesity was less complete. The findings confirm that RYGB does not indiscriminately lower body weight but specifically prevents excessive diet-induced obesity and ensuing metabolic impairments. This prevention of obesity model should be crucial for identifying the molecular mechanisms underlying gastric bypass surgery.

Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus.

Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS-expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS-immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα-immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFPVglut2 , EYFPVgat , and GFPGad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.

Galanin-Expressing GABA Neurons in the Lateral Hypothalamus Modulate Food Reward and Noncompulsive Locomotion.

The lateral hypothalamus (LHA) integrates reward and appetitive behavior and is composed of many overlapping neuronal populations. Recent studies associated LHA GABAergic neurons (LHA GABA ), which densely innervate the ventral tegmental area (VTA), with modulation of food reward and consumption; yet, LHA GABA projections to the VTA exclusively modulated food consumption, not reward. We identified a subpopulation of LHA GABA neurons that coexpress the neuropeptide galanin (LHA Gal ). These LHA Gal neurons also modulate food reward, but lack direct VTA innervation. We hypothesized that LHA Gal neurons may represent a subpopulation of LHA GABA neurons that mediates food reward independent of direct VTA innervation. We used chemogenetic activation of LHA Gal or LHA GABA neurons in mice to compare their role in feeding behavior. We further analyzed locomotor behavior to understand how differential VTA connectivity and transmitter release in these LHA neurons influences this behavior. LHA Gal or LHA GABA neuronal activation both increased operant food-seeking behavior, but only activation of LHA GABA neurons increased overall chow consumption. Additionally, LHA Gal or LHA GABA neuronal activation similarly induced locomotor activity, but with striking differences in modality. Activation of LHA GABA neurons induced compulsive-like locomotor behavior; while LHA Gal neurons induced locomotor activity without compulsivity. Thus, LHA Gal neurons define a subpopulation of LHA GABA neurons without direct VTA innervation that mediate noncompulsive food-seeking behavior. We speculate that the striking difference in compulsive-like locomotor behavior is also based on differential VTA innervation. The downstream neural network responsible for this behavior and a potential role for galanin as neuromodulator remains to be identified.SIGNIFICANCE STATEMENT The lateral hypothalamus (LHA) regulates motivated feeding behavior via GABAergic LHA neurons. The molecular identity of LHA GABA neurons is heterogeneous and largely undefined. Here we introduce LHA Gal neurons as a subset of LHA GABA neurons that lack direct innervation of the ventral tegmental area (VTA). LHA Gal neurons are sufficient to drive motivated feeding and locomotor activity similar to LHA GABA neurons, but without inducing compulsive-like behaviors, which we propose to require direct VTA innervation. Our study integrates galanin-expressing LHA neurons into our current understanding of the neuronal circuits and molecular mechanisms of the LHA that contribute to motivated feeding behaviors.

Neural Control of Energy Expenditure.

The continuous rise in obesity is a major concern for future healthcare management. Many strategies to control body weight focus on a permanent modification of food intake with limited success in the long term. Metabolism or energy expenditure is the other side of the coin for the regulation of body weight, and strategies to enhance energy expenditure are a current focus for obesity treatment, especially since the (re)-discovery of the energy depleting brown adipose tissue in adult humans. Conversely, several human illnesses like neurodegenerative diseases, cancer, or autoimmune deficiency syndrome suffer from increased energy expenditure and severe weight loss. Thus, strategies to modulate energy expenditure to target weight gain or loss would improve life expectancies and quality of life in many human patients. The aim of this book chapter is to give an overview of our current understanding and recent progress in energy expenditure control with specific emphasis on central control mechanisms.

Real-time monitoring of intracellular cAMP during acute ethanol exposure.

BACKGROUND: In previous studies, we have shown that ethanol (EtOH) enhances the activity of stimulatory G protein (Gs)-stimulated membrane-bound adenylyl cyclase (AC). The effect is AC isoform specific, and the type 7 AC (AC7) is most responsive to EtOH. In this study, we employed a fluorescence resonance energy transfer (FRET)-based cyclic AMP (cAMP) sensor, Epac1-camps, to examine real-time temporal dynamics of EtOH effects on cAMP concentrations. To our knowledge, this is the first report on real-time detection of the EtOH effect on intracellular cAMP. METHODS: Hela cells were transfected with Epac1-camps, dopamine (DA) receptor D1a , and 1 isoform of AC (AC7 or AC3). Fluorescent images were captured using a specific filter set for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and FRET, respectively, and FRET intensity was calculated on a pixel-by-pixel basis to examine changes in cAMP. RESULTS: During 2-minute stimulation with DA, the cytoplasmic cAMP level quickly increased and then decreased to a plateau, where the cAMP level was higher than the level prior to stimulation with DA. EtOH concentration dependently increased cytoplasmic cAMP in cells transfected with AC7, while EtOH did not have effect on cells transfected with AC3. Similar trends were observed for cAMP at the plasma membrane and in the nucleus during 2-minute stimulation with DA. Unexpectedly, when cells expressing AC7 were stimulated with DA or other Gs-coupled receptor's ligand plus EtOH for 5 seconds, EtOH reduced cAMP concentration. CONCLUSIONS: These results suggest that EtOH has 2 opposing effects on the cAMP-generating system in an AC isoform-specific manner, the enhancing effect on AC activity and the short-lived inhibitory effect. Thus, EtOH may have a different effect on cAMP depending on not only AC isoform but also the duration of exposure.

Effects of alcohols on recombinant adenylyl cyclase type 7 expressed in bacteria.

BACKGROUND: Our previous studies showed that ethanol enhanced the activity of adenylyl cyclase (AC) in an isoform-specific manner and that alcohol cutoff point of AC was isoform specific. Recently, we showed that 2,3-butanediol inhibited AC type 7 (AC7) activity in a stereoisomer-specific manner and that this inhibition was also AC isoform specific. These observations strongly suggest that a major target of alcohol action on cAMP signaling is AC. We hypothesized that alcohols exhibit their effect on AC activity by direct interaction with AC proteins. However, experimental systems employed in past studies such as intact cells and membrane preparations are too complex and do not allow us to unequivocally test this hypothesis. In attempt to bypass, these complications of the membrane-bound AC, we decided to study the effect of alcohols on AC recombinant proteins expressed in bacteria. METHODS: A recombinant AC, designated as AC7sol, consisting of the C(1a) and C(2) domains of the human AC7 was designed and expressed in bacteria. The activity of AC7sol was examined using lysate prepared from bacteria expressing AC7sol. RESULTS: The activity of AC7sol was stimulated by manganese or by the α subunit of G protein that stimulates AC (G(sα) ). Forskolin by itself did not stimulate the activity of AC7sol. However, in the presence of activated G(sα) , forskolin stimulated the activity of AC7sol. A series of n-alkanols including ethanol enhanced the manganese-stimulated activity of AC7sol. The alcohol cutoff point of AC7sol was pentanol. Ethanol and butanol increased V(max) and K(M) values of AC7sol. CONCLUSIONS: These results are consistent with our hypothesis and suggest that the enhancing effect of alcohols on AC activity is because of the increase in turnover number of AC. The current study demonstrates for the first time that the effect of alcohols requires only the C(1a) and C(2) domains of AC and no other domains of AC as well as no other mammalian proteins.

Vagally mediated effects of glucagon-like peptide 1: in vitro and in vivo gastric actions.

Glucagon-like peptide-1 (GLP-1) is a neuropeptide released following meal ingestion that, among other effects, decreases gastric tone and motility. The central targets and mechanism of action of GLP-1 on gastric neurocircuits have not, however, been fully investigated. A high density of GLP-1 containing neurones and receptors are present in brainstem vagal circuits, suggesting that the gastroinhibition may be vagally mediated. We aimed to investigate: (1) the response of identified gastric-projecting neurones of the dorsal motor nucleus of the vagus (DMV) to GLP-1 and its analogues; (2) the effects of brainstem application of GLP-1 on gastric tone; and (3) the vagal pathway utilized by GLP-1 to induce gastroinhibition. We conducted our experiments using whole-cell recordings from identified gastric-projecting DMV neurones and microinjection in the dorsal vagal complex (DVC) of anaesthetized rats while monitoring gastric tone. Perfusion with GLP-1 induced a concentration-dependent excitation of a subpopulation of gastric-projecting DMV neurones. The GLP-1 effects were mimicked by exendin-4 and antagonized by exendin-9-39. In an anaesthetized rat preparation, application of exendin-4 to the DVC decreased gastric tone in a concentration-dependent manner. The gastroinhibitory effects of exendin-4 were unaffected by systemic pretreatment with the pro-motility muscarinic agonist bethanechol, but were abolished by systemic administration of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), or by bilateral vagotomy. Our data indicate that GLP-1 activates selective receptors to excite DMV neurones mainly and that the gastroinhibition observed following application of GLP-1 in the DVC is due to the activation of an inhibitory non-adrenergic, non-cholinergic input to the stomach.